Sphingolipids (SL) comprise a group of lipids having ceramide, i.e., N-acylsphingosine as the basic group. There are two main types of SL, phosphoSL and glycoSL. While the former have one main component, i.e., sphingomyelin (ceramide-phosphorylcholine), the glycosphingolipids comprise a wide group. They range from monohexosylceramides (ceramide xcex2-glucose and ceramide xcex2-galactose), through oligohexosyl ceramide (e.g., di- and trihexosyl ceramides) to a large number of gangliosides composed of oligohexosyl ceramides to which sialic acid is also linked. SLs are present in practically every cell type and tissue and particularly abound in the nervous system. The relative composition of the SL may change with age; thus, it has been shown that the ratio of sphingomyelin to lecithin increases with age.
Glycosphingolipids have a high binding potential and act as specific receptors for a number of external agents, e.g., lectins, toxins, hormones and viruses. To exemplify: vibrio cholerae toxin links to GMI-ganglioside and Shigella dysenteriae verotoxins to globotriaosyl ceramide.
During the past decade there has been an enormous increase in research on sphingolipids due to discoveries that implicated members of this group in signal transduction processes [recently reviewed in Levade et al., Biochim. Biophys. Acta 1438, 1-17 (1999); Mathias et al., Biochem. J. 335, 465-480 (1998); Perry et al., Biochim. Biophys. Acta 1436, 233-243 (1998); Riboni et al., Prog. Lipid Res. 36, 153-195 (1997)]. The most studied compound was ceramide which was shown to play a role in the regulation of key processes such as growth inhibition, differentiation and apoptosis [Hannun et al., Biochim. Biophys. Acta 1154, 223-236; Hannun et al., Trends Cell Biol. 10, 73-80 (2001); Higgins et al., Trends Biochem. Sci. 17, 18-21 (1992)]. SPM is generally considered as the primary metabolic source of ceramide whose generation in a particular location in the cell, (e.g., the membrane) makes it suitable for mediating cellular signaling processes. An increased de novo synthesis of ceramide has also been described as a potential source for signaling [Bose et al., Cell 82, 405-414, (1995)]. Therefore, a major effort has been directed to modulate the generation of intracellular ceramide by sphingomyelinases, mostly the neutral, membrane-bound enzyme, although the acidic enzyme has also been implicated. Nevertheless, it should be emphasized that modification of the biosynthetic mechanisms such as reduction of the conversion of ceramide to SPM or glycolipids and, in parallel, its hydrolysis by ceramidases would also increase its concentration in the cell.
The role of sphingolipids in signal transduction [reviewed in L. Riboni et al., Prog. Lipid Res. 36, 153-195 (1997) and A. Gomez-Munoz, Biochim. Biophys. Acta 1391, 32-109 (1998)] have been extensively studied, and was proposed to operate through the xe2x80x9csphingomyelin cyclexe2x80x9d. According to this hypothesis, binding a particular extracellular ligand to its receptor activates a plasma membrane-bound sphingomyelinase, giving rise to ceramide, which acts as a mediator of the intracellular effects of the ligand. Numerous publications describe and emphasize the role of ceramide in cell killing by apoptosis as well as its effect on important cellular events such as proliferation, differentiation and reaction to stress conditions. Of particular interest are also reports that short chain, cell-permeable (e.g., C2 or C6) ceramides evoke biological responses that lead to cell killing. Other studies, using the precursor of ceramide, i.e., sphingosine have shown its effects on cell growth and viability. Furthermore, sphingosine was shown to inhibit protein kinase C and increase the intracellular concentration of calcium ions. The phosphorylated form of sphingosine, i.e., sphingosine-1-phosphate has been shown to be a potent activator of phospholipase D. And di- or tri-methylated sphingosine was shown to inhibit growth of cancer cells [Endo et al., Cancer Research 51, 1613-1618, (1981)].
The involvement of ceramide and sphingolipid metabolism in cancer has been studied. It have been demonstrated that apoptosis induced by administration of a variety of chemotherapeutic agents is mediated by ceramide [Strum et al., J. Biol. Chem. 269, 15493-15497 (1994); Maurer et al., J. Natl. Cancer Inst. 91, 1138-1146 (1999); Suzuki et al., Exp. Cell Res. 233, 41-47 (1997)]. Anthracyclins (e.g., daunorubicin) have been shown to induce ceramide accumulation which subsequently led to death of cancer cells [Bose et al., Cell 82, 405-414 (1995)]. The second line of study showed that drug-resistant cancer cells differ in their sphingolipid metabolism from drug-sensitive ones. Of special interest in this respect are studies of Cabot et al. [Lavie et al., J. Biol. Chem. 271, 19530-19536 (1996)], who have demonstrated that glucosylceramide, a direct metabolic product of ceramide, was elevated in several drug-resistant cells overexpressing the P-glycoprotein pump (Pgp). Overexpression of the enzyme that synthesizes this glycolipid, i.e., glucosylceramide synthetase (GCS), by a retroviral expression system resulted in conversion of doxorubicin-sensitive cells into resistant ones [Liu et al., J. Biol. Chem. 274, 1140-1146 (1999)]. Conversely, inhibition of GCS expression, by antisense technology, resulted in increased sensitivity to doxorubicin. Cabot et al. have also proposed that drug-resistance modulators such as tamoxifen, verapamil and the cyclosporine analog, PSC 833, exert their effect by inhibition of GCS [Cabot et al., FEBS Letters 394, 129-131 (1996), FEBS Letters 431, 185-199 (1998); Lavie et al., J. Biol. Chem. 272, 1682-1687 (1999); Lucci et al., Cancer 86, 300-311 (1999)], resulting in an increase of cellular ceramide. Nicholson et al. (British J. Cancer 81, 423-430 (1989)] have shown that an inhibitor of GCS, 1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol, killed preferentially multidrug-resistant cells, compared to their drug-sensitive counterparts. Taken together, the above studies suggest a metabolic mechanism which in MDR-cells decrease their ceramide content by converting it to glucosylceramide, making them resistant to a series of chemotherapeutic drugs.
Of special interest is the mechanism proposed for the anticancer drug hexadecylphosphocholine [HePC, Wieder et al., J. Biol. Chem. 273, 11025-11031, (1998)]. This is an antiproliferative drug, which is currently used for the treatment of extraneous metastases of mammary carcinoma and has been shown to induce apoptosis at a concentration of 25 xcexcM. The above publication provides support that HePC, which inhibits the biosynthesis of phosphatidylcholine exerts a secondary effect by decreasing the biosynthesis of sphingomyelin and consequently increasing the levels of ceramide and it is probably the latter that is responsible for the proapoptotic properties of HePC. And, indeed the authors showed that the PC-induced apoptosis was blocked by Fumonisin B1, an inhibitor of ceramide synthesis. And, short-chain, membrane-permeable ceramides additively increased the apoptotic effect of HePC.
Another major aspect of the metabolism of the sphingolipids is their accumulation in organs of patients afflicted with the genetic lipid storage diseases, such as Gaucher disease (xcex2-glucosidase), Tay-Sachs disease (xcex2-N-acetyl hexosaminidase); Niemann-Pick disease (acid sphingomyelinase), Krabbe disease (xcex2-galactosidase), Metachromatic leukodystrophy (arylsulfatase A), Fabry disease (ceramidase) and Farber disease (xcex1-galactosidase). Each of these diseases is due to a mutation in a gene encoding a lysosomal sphingolipid hydrolase (shown in brackets). Consequently, the activity of the respective hydrolase is considerably reduced resulting in accumulation of the respective sphingolipid in the patients"" organs.
Being a metabolic disorder, the metabolic defect and accumulation of the corresponding sphingolipid is a life-long phenomenon. Three forms of therapy are being used or considered. 1. Enzyme replacement therapy, in which the enzyme involved is purified and infused into the patients for the rest of their lives; this approach is currently applied to patients with Gaucher disease, in which the patients are infused with xcexc-glucosidase purified from human placentae or, alternatively, a recombinant form of the enzyme. 2. Gene therapy, in which a gene encoding the normal enzyme will be cloned and administered into the patients; this is currently in the stage of planning. 3. Infusion into the patient of an inhibitor of the biosynthesis of the sphingolipid accumulating in the disease, the aim being to reduce the quantity of the sphingolipid accumulating in the patients"" organs. This approach is currently in clinical test, on Gaucher disease patients in several medical centers [Cox et al., The Lancet 355, 1481-1485, (2000)].
Sphingolipids are of the general structure:
CH3(CH2)12xe2x80x94CHxe2x95x90CHxe2x80x94CHOHxe2x80x94CHNH[COR1]xe2x80x94CH2OR2 
wherein R1 is CH3(CH2)14-22 and R2 may be a hydrogen atom, phosphoryl-choline; glucose, galactose or an oligosaccharide.
Ceramide, in which R2 is hydrogen, the precursor of the sphingolipids, is a bioeffector molecule, affecting cell differentiation, apoptosis and growth suppression.
Several non-natural analogs of ceramide have been synthesized having a phenyl group instead of the CH3(CH2)12xe2x80x94CHxe2x95x90CH residue.
For example, the compound PDMP 
has been shown to be an inhibitor of glucosphingolipid [Vunnam and Radin Chem. Phys. Lipid 26, 265 (1980)].
Acyl phenyl amino alcohol (MAPP): 
has been shown to inhibit ceramidase, resulting in an inhibition of cell growth [Bielawska et al., J. Biol. Chem. 271, 12646-12654 (1996)].
Esters of p-nitrophenyl-amino-propanediol: 
have been shown to inhibit cell differentiation. [Bielawska et al., J. Biol. Chem. 267, 18493-18497 (1992)].
Other non-natural derivatives of sphinglipids affect cell growth and differentiation. For example N,N,N-trimethyl sphingosine has been shown to inhibit cell growth [Endo et al., Cancer Research, 51, 1613-1618 (1991)]. C8 ceramide in which the amide group was replaced by xe2x80x94NHxe2x80x94(CH2)7CH3: CH3(CH2)12xe2x80x94CHxe2x95x90CHxe2x80x94CHOHxe2x80x94CHNH[(CH2)7CH3]xe2x80x94CH2OH induced apoptosis [Karasavvas et al., Eur. J Biochem. 236, 729-731 (1996)].
Hexadecylphosphocholine induced a ceramide-mediated apoptosis [Wieder et al., J. Biol. Chem. 273, 11025-11031 (1998)].
It is an object of the present invention to provide novel therapeutic compounds that can modify the metabolism of sphingolipids.
It is a further object of this invention to provide novel therapeutic compounds that may be used for killing of unwanted cells.
These and other objects of the invention will become clearer as the description proceeds.
The invention relates to compounds of the general formula (1): 
wherein
R represent a linear or branched, saturated, or unsaturated alkyl or alkenyl chain, which may optionally be substituted by hydroxyl, CH3(CH2)mCHxe2x95x90CHxe2x80x94, CH3(CH2)m, wherein m is zero or an integer of from 1 to 20, phenyl, optionally substituted by nitro, amino, alkylamino, acylamino, xe2x80x94NHC(S)NH-alkyl, sulfonylamido-alkyl, a group 
xe2x80x83wherein n is an integer of from 1 to 20, or a group xe2x80x94NH-adamantane, xe2x80x94NH-t-BOC, xe2x80x94NH-FMOC or NH-CBZ;
X represents hydrogen or the group xe2x80x94OR4 in which R4 is hydrogen or a linear or branched, saturated or unsaturated C1-C6 alkyl or alkenyl chain which may be optionally substituted with hydroxy;
Y represents xe2x80x94NH2, NHRx wherein Rx is hydrogen, a linear or branched alkyl or alkenyl chain which may be optionally substituted with hydroxy, an amino protecting group, 
xe2x80x83xe2x80x94NH(SO2)R1, xe2x80x94NR1R2, xe2x80x94N+R1R2R3, wherein R1, R2 and R3, which may be identical or different each represent C1-6alkyl or C1-6alkenyl, a group 
xe2x80x83wherein n is zero or an integer of from 1 to 20, a group xe2x80x94NH-adamantane, a group 
xe2x80x83where xe2x80x9cpolymerxe2x80x9d designates a natural or synthetic biocompatible polymer having a molecular weight between 103 and 106 daltons;
Z represents hydrogen, xe2x80x94OH, a mono- or disaccharide, a monosaccharide sulfate and choline phosphate;
xe2x80x83with the proviso that
Y cannot represent NH2 when R represents an alkyl, the group CH3(CH2)mCHxe2x95x90CHxe2x80x94, phenyl or nitro phenyl; and
xe2x80x83Y cannot represent the groups xe2x80x94NR1R2 or xe2x80x94N+R1R2R3, or NHR4 where R4 represents octyl when R1 represents a methyl, R represents the group CH3(CH2)mCHxe2x95x90CHxe2x80x94 and Z represents xe2x80x94OH;
xe2x80x83and isomers and pharmaceutically acceptable salts thereof.
The invention also relates to a pharmaceutical composition comprising as active ingredient a compound of formula (I) wherein the substituents are as defined in claim 1, and optionally further comprising pharmaceutically acceptable carrier, adjuvant or diluent.
The pharmaceutical compositions of the invention may be used for reducing accumulation of sphinglipids, and thus for the treatment of lipid storage diseases such as Gaucher disease, Tay-Sachs disease, Niemann-Pick disease, Krabbe disease, Metachromatic leukodystrophy, Fabry disease and Farber disease.
The novel compounds of formula (I) may be used as inhibitors of acidic, neutral and alkaline sphingomyelinases, acidic, neutral and alkaline ceramidases, xcex1-galactosyl synthetase, ceramide synthetase, sphingomyelin synthetase and glycoceramides synthetase.
The pharmaceutical compositions of the invention may also be used for the treatment of cancerous diseases, for killing of wild type and drug-resistant cancer cells.
The pharmaceutical compositions of the invention may also be used for the treatment of parasitic, viral, bacterial, fungal and prion diseases.
In a further aspect the invention relates to a method of treating a lipid storage disease or a cancerous disease in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a compound of formula (I) or of pharmaceutical composition comprising the same.